Loss of mouse cardiomyocyte talin-1 and talin-2 leads to β-1 integrin reduction, costameric instability, and dilated cardiomyopathy

Abstract

Continuous contraction-relaxation cycles of the heart require strong and stable connections of cardiac myocytes (CMs) with the extracellular matrix (ECM) to preserve sarcolemmal integrity. CM attachment to the ECM is mediated by integrin complexes localized at the muscle adhesion sites termed costameres. The ubiquitously expressed cytoskeletal protein talin (Tln) is a component of muscle costameres that links integrins ultimately to the sarcomere. There are two talin genes, Tln1 and Tln2. Here, we tested the function of these two Tln forms in myocardium where Tln2 is the dominant isoform in postnatal CMs. Surprisingly, global deletion of Tln2 in mice caused no structural or functional changes in heart, presumably because CM Tln1 became up-regulated. Tln2 loss increased integrin activation, although levels of the muscle-specific β1D-integrin isoform were reduced by 50%. With this result, we produced mice that had simultaneous loss of both CM Tln1 and Tln2 and found that cardiac dysfunction occurred by 4 wk with 100% mortality by 6 mo. β1D integrin and other costameric proteins were lost from the CMs, and membrane integrity was compromised. Given that integrin protein reduction occurred with Tln loss, rescue of the phenotype was attempted through transgenic integrin overexpression, but this could not restore WT CM integrin levels nor improve heart function. Our results show that CM Tln2 is essential for proper β1D-integrin expression and that Tln1 can substitute for Tln2 in preserving heart function, but that loss of all Tln forms from the heart-muscle cell leads to myocyte instability and a dilated cardiomyopathy.

Keywords: cardiomyopathy; costameres; heart; integrins; talin.

Fig. 1.

Tln2KO shows no alterations in myocardial structure, hypertrophic markers, or whole-heart Tln1 expression. (A) Histological analyses showed no difference between WT and Tln2KO male mice at 1 y of age. [Hematoxylin/eosin staining (H&E) was used to display standard morphology; trichrome was used to detect fibrotic alterations.] (B) qRT-PCR was used to detect changes in the molecular markers of hypertrophy, ANF, BNP, and α-skeletal actin transcripts, using heart RNA from WT and Tln2KO male mice at 6 mo. GAPDH was used as loading control. Data are mean ± SEM; n = 3 in each group. (C) Western blotting of whole-heart protein lysates from 2-mo-old Tln2KO vs. WT shows absence of Tln2 protein but no changes in Tln1. GAPDH was used as loading control. WT: n = 7; Tln2KO: n = 5. (D) Dual staining was performed with anti-Tln2 (green) and anti-Dys (red), used as a myocyte membrane marker, on adult cardiac tissue from WT and Tln2KO male mice. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 2.

Tln1 can functionally replace Tln2 in cardiac myocytes. (A and B) Western blotting (A) and associated densitometric analysis (B) of isolated cardiomyocyte lysate from 2-mo-old Tln2KO vs. WT. Data are mean ± SEM; n = 7 in each group. (**P < 0.01.) (C) qRT-PCR was used to quantify Tln1 in isolated CM from WT and Tln2KO male mice. Data are mean ± SEM; n = 3 in each group. Data were analyzed by t test vs. WT and were significant as indicated (**P < 0.01). (D) Immunofluorescent microscopy of WT and Tln2KO adult cardiac tissue shows strong localization of Tln1 at costameres of Tln2KO CM. Dys was used to mark and localize cardiac myocytes. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 3.

Integrins and integrin complex proteins are altered in Tln2KO CM. (A and B) Western blotting (A) and densitometric analysis (B) of isolated cardiac myocytes from 2-mo-old WT and Tln2KO male mice show reduced β1D integrin, ILK, and Pax expression in the Tln2KO without changes in other proteins assessed—α7 integrin, Vcl, KN2, or dystrophin. GAPDH was used as a loading control. Data are mean ± SEM; n = 7 in each group. (C) β1D-integrin transcript expression was not changed in the Tln2KO examined by qRT-PCR analysis of RNA from WT and Tln2KO male mice isolated myocytes. Data are mean ± SEM; n = 3 in each group. (D) β1 integrin was visibly reduced, but normally localized in Tln2KO myocytes when compared with WT myocardium. (E) Vcl levels and localization remained unchanged when assessed by immunofluorescent microscopy of adult cardiac tissue from WT and Tln2KO male mice. (**P < 0.01 and ***P < 0.001: Tln2KO vs. WT.) ITG complex, integrin complex. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 4.

β1 activation in cardiac myocytes is increased in Tln2KO myocardium. (A) Immunofluorescent microscopy using antibody 9EG7 to detect β1-integrin activation and Dys to detect myocytes in adult cardiac tissue from WT and Tln2KO male mice. (B) Western blotting and densitometric analysis for activated β1 integrin (9EG7) and total β1D integrin in isolated cardiac myocytes from 2-mo-old WT and Tln2KO male mice. GAPDH was used as loading control. Data are mean ± SEM; n = 7 in each group. Data were analyzed by t test vs. WT and were significant as indicated (*P < 0.05). A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 5.

Combined loss of Tln1 and Tln2 from CMs leads to a dilated cardiomyopathy and reduced survival. (A) Survival curve shows that Tln1cKO/Tln2KO male mice die between 2 and 6 mo of age [Tln1flox/flox/Tln2KO (control): n = 23; Tln1cKO/Tln2KO: n = 16]. (B) Morphometric data of male mice that survived to 8 wk showed increased HW/BW and HW/TL in the basal Tln1cKO/Tln2KO vs. Tln1flox/flox/Tln2KO used as controls. Data are mean ± SEM, Tln1flox/flox/Tln2KO n = 7 and Tln1cKO/Tln2KO n = 10. Data were analyzed by t test vs. Tln1flox/flox/Tln2KO and were significant as indicated (*P < 0.05; **P < 0.01). (C) Histological (H&E and trichrome staining) analyses showed cardiac dilation with fibrosis in 8-wk-old male Tln1cKO/Tln2KO mice vs. control Tln1flox/flox/Tln2KO. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 6.

Loss of all Tln forms from the CM leads to concomitant reduction of CM integrin and costameric proteins. Immunofluorescent microscopy of Tln1flox/flox/Tln2KO and Tln1cKO/Tln2KO cardiac tissue from 4-wk-old male mice for (A) Tln1 and β1 integrin (β1) and for (B) Vcl and Dys shows that, when all Tln isoforms in CMs are deleted, neither β1 integrin nor Vcl are highly localized at the CM membrane. DAPI (blue) was used to identify nuclei. Asterisks indicate CMs from Tln1cKO/Tln2KO hearts in which Tln1 expression remains, as is common given the chimeric expression of Cre transgenes. These Tln1 expressing CMs still show β1 integrin and Vcl expression at the membrane. Arrows indicate non-CMs in Tln1cKO/Tln2KO hearts. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 7.

CM membrane stability is lost in the Tln-deficient cell. EBD was used to assess for membrane permeability, indicative of early cell damage. Tln1flox/flox/Tln2KO and Tln1cKO/Tln2KO mice were injected i.p. with EBD at 4 wk, and heart tissue was analyzed for EBD localization, shown as red. Anti-Dys antibody was used to identify myocyte cell membranes. Significant EBD uptake was only detected in the Tln1cKO/Tln2KO hearts. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 8.

Loss of Tln from cultured NMVMs recapitulates the in vivo findings, showing that talin protein is required for preservation of normal β1-integrin expression, costamere structure, and sarcomere organization. Immunofluorescent microscopy of Tln1flox/flox/Tln2KO NMVMs infected with Ad/LacZ (control) and Ad/Cre (to excise the floxed Tln1 gene) for 72 h for (A) Tln1 (green) and β1D (red), (B) vinculin (Vcl, green) and Phalloidin (Phall, red), and (C) focal adhesion kinase (FAK, red) and s–α-act (green). DAPI (blue) was used to localize nuclei. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 9.

α7β1D integrin overexpression in Tln-deficient mice does not rescue the cardiomyopathy or prevent premature death observed in Tln1cKO/Tln2KO. (A) Lysates from Tln1flox/flox/Tln2KO, Tln1cKO/Tln2KO, Tln1flox/flox/Tln2KO/α7β1D OE, and Tln1cKO/Tln2KO/α7β1D OE adult cardiac tissue (8 wk) were analyzed by Western blotting for β1D and α7 integrins. GAPDH was used as a loading control. (B) Histological (H&E and trichrome staining) analyses showed cardiac dilation with fibrosis in 8-wk-old male Tln1cKO/Tln2KO/α7β1D OE mice compared with Tln1flox/flox/Tln2KO/α7β1D OE. (C) Echocardiographic analysis. Cardiac function (%FS) and wall thickness (LVPwd and IVSd) were decreased, whereas left ventricle (LV) dilation occurred (LVID) in Tln1cKO/Tln2KO and Tln1cKO/Tln2KO/α7β1D OE mice compared with Tln1flox/flox/Tln2KO and Tln1flox/flox/Tln2KO /α7β1D OE mice (Tln1flox/flox/Tln2KO: n = 4; Tln1cKO/Tln2KO: n = 8; Tln1flox/flox/Tln2KO/α7β1D OE: n = 4; Tln1cKO/Tln2KO/α7β1D OE: n = 4, all at 8 wk). (D) Morphometry in 8-wk-old male mice (Tln1flox/flox/Tln2KO: n = 10; Tln1cKO/Tln2KO: n = 7; Tln1flox/flox/Tln2KO/α7β1D OE: n = 7; Tln1cKO/Tln2KO/α7β1D OE: n = 6). Data in A and B are mean ± SEM. Data were analyzed by t tests and were significant as indicated (Tln1flox/flox/Tln2KO vs. Tln1cKO/Tln2KO: *P < 0.05; **P < 0.01; ***P < 0.001; Tln1flox/flox/Tln2KO/α7β1D OE vs. Tln1cKO/Tln2KO/α7β1D OE: ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; Tln1cKO/Tln2KO vs. Tln1cKO/Tln2KO/α7β1D OE: ^{^}P < 0.05). FS: fractional shortening; IVSd: interventricular septum in diastole; LVPwd: left ventricular posterior wall in diastole; LVIDd: left ventricular internal dimension in diastole. (E) Immunofluorescent microscopy of Tln1cKO/Tln2KO/α7β1D cardiac tissue from 8-wk-old male mice for Tln1 (green) and β1D integrin (β1D) (red) shows that β1D integrin could not be located at the cellular membrane in Tln1-deficient CMs, even with transgenic CM-specific β1D overexpression. Asterisks indicate CMs in which Tln1 expression remains, as is common given the chimeric expression of Cre transgenes. β1D integrin expression is still detected at the membrane in these cells. Arrows indicate CMs with total Tln (Tln1 and Tln2) deletion. A higher-quality version of this figure can be viewed in the SI Appendix.

Fig. 10.

Forced overexpression of β1 integrins cannot be achieved in Tln-deficient NMVMs, although β1D integrin mRNA levels are restored toward normal. Tln1flox/flox/Tln2KO NMVMs were infected with human Ad/β1D or Ad/α5 for 48 h and subsequently with Ad/LacZ or Ad/Cre for an additional 72 h. (A) Protein lysates were analyzed by Western blotting for Tln1, β1D, α5, Vcl, and Pax. GAPDH and α-tubulin were used as loading controls. (B) Phase-contrast images of cell groups. (C) Densitometric quantification of Western blot data from A analyzed for Tln1 and β1D. (D) Tln deletion from NMVM leads to increased LDH release, indicating cell injury, compared with Ad/LacZ-treated control cells. (E) qRT-PCR was used to quantify Tln1, endogenous β1D, and exogenous human β1D integrin. Data for all panels are mean ± SEM; n = 3. All data were analyzed by t tests vs. AdLacZ and were significant as indicated (*P < 0.05; **P < 0.01; ***P < 0.001). A higher-quality version of this figure can be viewed in the SI Appendix.